Proteome-wide detection of Abl1 SH3-binding peptides by integrating computational prediction and peptide microarray

Protein-protein interactions are essential for regulating almost all aspects of cellular functions. Many of these interactions are mediated by weak and transient protein domain-peptide binding, but they are often under-represented in high throughput screening of protein-protein interactions using techniques such as yeast two-hybrid and mass spectrometry. On the other hand, computational predictions and in vitro binding assays are valuable in providing clues of in vivo interactions. We present here a systematic approach that integrates computer modeling and a peptide microarray technology to identify binding peptides of the SH3 domain of the tyrosine kinase Abl1 in the human proteome. Our study provides a comprehensive list of candidate interacting partners for the Abl1 protein, among which the presence of numerous methyltransferases and RNA splicing proteins may suggest a novel function of Abl1 in chromatin remodeling and RNA processing. This study illustrates a powerful approach for integrating computational and experimental methods to detect protein interactions mediated by domain-peptide recognition.     

Cell-cell and cell-ECM interactions in epithelial apoptosis and cell renewal during frog intestinal development

Amphibian intestinal remodeling during metamorphosis is a developmental system that is entirely controlled by thyroid hormone. It transforms a simple tubular organ into a complex multiply folded frog intestine similar to that in higher vertebrates. This process involves the degeneration of the larval epithelium through programmed cell death (apoptosis) and concurrent proliferation and differentiation of adult cell types. Earlier morphological and cellular studies have provided strong evidence implicating the importance of cell-cell and cell-ECM (extracellular matrix) interactions in this process. The recent molecular characterization of the genes that are regulated by thyroid hormone has begun to reveal some molecular clues underlying such interactions. In particular, theXenopus putative morphogen hedgehog appears to be involved in regulating/mediating cell-cell interactions during adult epithelial proliferation, differentiation, and/or intestinal morphogenesis. On the other hand, several matrix metalloproteinases (MMPs) may be involved in remodeling the ECM. Of special interest is stromelysin-3, whose spatial and temporal expression profile during intestinal metamorphosis implicates a role in ECM remodeling, which in turn facilitates cell fate determination, i.e., apoptosis vs proliferation and differentiation. Understanding the mechanisms of action for those extracellular molecules will present a future challenge in developmental research.     

An abstract cell model that describes the self-organization of cell function in living systems

The principal aim of systems biology is to search for general principles that govern living systems. We develop an abstract dynamic model of a cell, rooted in Mesarovi? and Takahara's general systems theory. In this conceptual framework the function of the cell is delineated by the dynamic processes it can realize. We abstract basic cellular processes, i.e., metabolism, signalling, gene expression, into a mapping and consider cell functions, i.e., cell differentiation, proliferation, etc. as processes that determine the basic cellular processes that realize a particular cell function. We then postulate the existence of a 'coordination principle' that determines cell function. These ideas are condensed into a theorem: If basic cellular processes for the control and regulation of cell functions are present, then the coordination of cell functions is realized autonomously from within the system. Inspired by Robert Rosen's notion of closure to efficient causation, introduced as a necessary condition for a natural system to be an organism, we show that for a mathematical model of a self-organizing cell the associated category must be cartesian closed. Although the semantics of our cell model differ from Rosen's (M,R)-systems, the proof of our theorem supports (in parts) Rosen's argument that living cells have non-simulable properties. Whereas models that form cartesian closed categories can capture self-organization (which is a, if not the, fundamental property of living systems), conventional computer simulations of these models (such as virtual cells) cannot. Simulations can mimic living systems, but they are not like living systems.     

Ephrins and their receptors: binding versus biology

Ephrins and Eph receptors play important roles in the development of the central nervous system and peripheral tissues by orchestrating cellular movements, resulting in events such as axonal growth cone guidance, tissue segmentation, and angiogenic remodeling. To understand the role of specific ephrin and Eph receptor interactions, it is important to identify the binding specificity between individual ligand-receptor complexes. To date, a dogma in the field suggests that there may be promiscuous binding within the subclasses of the ephrin family. However, this overlooks and contradicts several binding studies that suggest specificity within each subclass. Although binding studies only provide evidence on the dynamics and strength of protein interactions, they do not indicate whether particular interactions are physiologically relevant. Thus, distribution and gene targeted mutations of ephrins and their receptors can provide critical insights into the relevance of specific ligand-receptors interactions. This review mainly focuses on the B-class family and will evaluate the differences between binding affinities and biological functions, importance of oligomeric interactions, and structural differences and similarities between classes.     

On the Potential of Machine Learning to Examine the Relationship Between Sequence,Structure, Dynamics and Function of Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) constitute a broad set of proteins with few uniting and many diverging properties. IDPs—and intrinsically disordered regions (IDRs) interspersed between folded domains—are generally characterized as having no persistent tertiary structure; instead they interconvert between a large number of different and often expanded structures. IDPs and IDRs are involved in an enormously wide range of biological functions and reveal novel mechanisms of interactions, and while they defy the common structure-function paradigm of folded proteins, their structural preferences and dynamics are important for their function. We here discuss open questions in the field of IDPs and IDRs, focusing on areas where machine learning and other computational methods play a role. We discuss computational methods aimed to predict transiently formed local and long-range structure, including methods for integrative structural biology. We discuss the many different ways in which IDPs and IDRs can bind to other molecules, both via short linear motifs, as well as in the formation of larger dynamic complexes such as biomolecular condensates. We discuss how experiments are providing insight into such complexes and may enable more accurate predictions. Finally, we discuss the role of IDPs in disease and how new methods are needed to interpret the mechanistic effects of genomic variants in IDPs.     

Mechanisms and context underlying the role of autophagy in cancer metastasis

Macroautophagy/autophagy is a fundamental cellular degradation mechanism that maintains cell homeostasis, regulates cell signaling, and promotes cell survival. Its role in promoting tumor cell survival in stress conditions is well characterized, and makes autophagy an attractive target for cancer therapy. Emerging research indicates that autophagy also influences cancer metastasis, which is the primary cause of cancer-associated mortality. However, data demonstrate that the regulatory role of autophagy in metastasis is multifaceted, and includes both metastasis-suppressing and -promoting functions. The metastasis-suppressing functions of autophagy, in particular, have important implications for autophagy-based treatments, as inhibition of autophagy may increase the risk of metastasis. In this review, we discuss the mechanisms and context underlying the role of autophagy in metastasis, which include autophagy-mediated regulation of focal adhesion dynamics, integrin signaling and trafficking, Rho GTPase-mediated cytoskeleton remodeling, anoikis resistance, extracellular matrix remodeling, epithelial-to-mesenchymal transition signaling, and tumor-stromal cell interactions. Through this, we aim to clarify the context-dependent nature of autophagy-mediated metastasis and provide direction for further research investigating the role of autophagy in cancer metastasis.     

Structural overview of toxin–antitoxin systems in infectious bacteria: A target for developing antimicrobial agents

The bacterial toxin–antitoxin (TA) system is a module that may play a role in cell survival under stress conditions. Generally, toxin molecules act as negative regulators in cell survival and antitoxin molecules as positive regulators. Thus, the expression levels and interactions between toxins and antitoxins should be systematically harmonized so that bacteria can escape such harmful conditions. Since TA systems are able to control the fate of bacteria, they are considered potent targets for the development of new antimicrobial agents. TA systems are widely prevalent with a variety of systems existing in bacteria: there are three types of bacterial TA systems depending on the property of the antitoxin which binds either the protein toxin or mRNA coding the toxin protein. Moreover, the multiplicity of TA genes has been observed even in species of bacteria. Therefore, knowledge on TA systems such as the individual characteristics of TA systems, integrative working mechanisms of various TA systems in bacteria, interactions between toxin molecules and cellular targets, and so on is currently limited due to their complexity. In this regard, it would be helpful to know the structural characteristics of TA modules for understanding TA systems in bacteria. Until now, 85 out of the total structures deposited in PDB have been bacterial TA system proteins including TA complexes or isolated toxins/antitoxins. Here, we summarized the structural information of TA systems and analyzed the structural characteristics of known TA modules from several bacteria, especially focusing on the TA modules of several infectious bacteria.     

The physical basis of microtubule structure and stability

Microtubules are cylindrical polymers found in every eukaryotic cell. They have a unique helical structure that has implications at both the cellular level, in terms of the functions they perform, and at the multicellular level, such as determining the left-right symmetry in plants. Through the combination of an atomically detailed model for a microtubule and large-scale computational techniques for computing electrostatic interactions, we are able to explain the observed microtubule structure. On the basis of the lateral interactions between protofilaments, we have determined that B lattice is the most favorable configuration. Further, we find that these lateral bonds are significantly weaker than the longitudinal bonds along protofilaments. This explains observations of microtubule disassembly and may serve as another step toward understanding the basis for dynamic instability.     

The first three minutes: smooth muscle contraction, cytoskeletal events, and soft glasses.

Smooth muscle exhibits biophysical characteristics and physiological behaviors that are not readily explained by present paradigms of cytoskeletal and cross-bridge mechanics. There is increasing evidence that contractile activation of the smooth muscle cell involves an array of cytoskeletal processes that extend beyond cross-bridge cycling and the sliding of thick and thin filaments. We review here the evidence suggesting that the biophysical and mechanical properties of the smooth muscle cell reflect the integrated interactions of an array of highly dynamic cytoskeletal processes that both react to and transform the dynamics of cross-bridge interactions over the course of the contraction cycle. The activation of the smooth muscle cell is proposed to trigger dynamic remodeling of the actin filament lattice within cellular microdomains in response to local mechanical and pharmacological events, enabling the cell to adapt to its external environment. As the contraction progresses, the cytoskeletal lattice stabilizes, solidifies, and forms a rigid structure well suited for transmission of tension generated by the interaction of myosin and actin. The integrated molecular transitions that occur within the contractile cycle are interpreted in the context of microscale agitation mechanisms and resulting remodeling events within the intracellular microenvironment. Such an interpretation suggests that the cytoskeleton may behave as a glassy substance whose mechanical function is governed by an effective temperature.     

Dynamic imaging of cellular interactions with extracellular matrix

Adhesive and proteolytic interactions of cells with components of the extracellular matrix (ECM) are fundamental to morphogenesis, tissue assembly and remodeling, and cell migration as well as signal acquisition from tissue-bound factors. The visualization from fixed samples provides snapshot-like, static information on the cellular and molecular dynamics of adhesion receptor and protease functions toward ECM, such as interstitial fibrillar tissues and basement membranes. Recent technological developments additionally support the dynamic imaging of ECM scaffolds and the interaction behavior of cells contained therein. These include differential interference contrast, confocal reflection microscopy, optical coherence tomography, and multiphoton microscopy and second-harmonic generation imaging. Most of these approaches are combined with fluorescence imaging using derivates of GFP and/or other fluorescent dyes. Dynamic 3D imaging has revealed an unexpected degree of dynamics and turnover of cell adhesion and migration as well as basic mechanisms that lead to proteolytic remodeling of connective tissue by stromal cells and invading tumor cells.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0682-0The Histochemistry and Cell Biology Lecture presented at the 12th International Congress of Histochemistry and Cytochemistry in La Jolla, California, USA, 24–28 July 2004     

Signaling networks regulating leukocyte podosome dynamics and function

Podosomes are ventral adhesion structures prominent in cells of the myeloid lineage. A common aspect of these cells is that they are highly motile and must to traverse multiple tissue barriers in order to perform their functions. Recently podosomes have gathered attention from researchers as important cellular structures that can influence cell adhesion, motility and matrix remodeling. Adhesive and soluble ligands act via transmembrane receptors and propagate signals to the leukocyte cytoskeleton via small G proteins of the Rho family, tyrosine kinases and scaffold proteins and are able to induce podosome formation and rearrangements. Manipulation of the signals that regulate podosome formation and dynamics can therefore be a strategy to interfere with leukocyte functions in a multitude of pathological settings, such as infections, atherosclerosis and arthritis. Here, we review the major signaling molecules that act in the formation and regulation of podosomes.     

Extracellular Matrix Degradation and Remodeling in Development and Disease

The extracellular matrix (ECM) serves diverse functions and is a major component of the cellular microenvironment. The ECM is a highly dynamic structure, constantly undergoing a remodeling process where ECM components are deposited, degraded, or otherwise modified. ECM dynamics are indispensible during restructuring of tissue architecture. ECM remodeling is an important mechanism whereby cell differentiation can be regulated, including processes such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair. In contrast, abnormal ECM dynamics lead to deregulated cell proliferation and invasion, failure of cell death, and loss of cell differentiation, resulting in congenital defects and pathological processes including tissue fibrosis and cancer. Understanding the mechanisms of ECM remodeling and its regulation, therefore, is essential for developing new therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine.The extracellular matrix (ECM) forms a milieu surrounding cells that reciprocally influences cellular function to modulate diverse fundamental aspects of cell biology (Hynes 2009). The diversity and sophistication of ECM components and their respective cell surface receptors are among the most salient features during metazoan evolution (Har-el and Tanzer 1993; Hutter et al. 2000; Whittaker et al. 2006; Engler et al. 2009; Huxley-Jones et al. 2009; Ozbek et al. 2010). The ECM is extremely versatile and performs many functions in addition to its structural role. As a major component of the microenvironment of a cell, the ECM takes part in most basic cell behaviors, from cell proliferation, adhesion and migration, to cell differentiation and cell death (Hynes 2009). This pleiotropic aspect of ECM function depends on the highly dynamic structure of ECM and its remodeling as an effective mechanism whereby diverse cellular behaviors can be regulated. This concept is particularly important when considering processes and cell behaviors that need to be deployed promptly and transiently and wherein cell–cell and cell–matrix interactions are constantly changing (Daley et al. 2008).ECM dynamics are a feature of tissues wherein radical remodeling occurs, such as during metamorphosis of insects and amphibians or remodeling of the adult bone and mammary gland, and in developmental processes, including neural crest migration, angiogenesis, tooth and skeletal development, branching morphogenesis, maturation of synapses, and the nervous system (Berardi et al. 2004; Fukumoto and Yamada 2005; Page-McCaw et al. 2007; Zimmermann and Dours-Zimmermann 2008).ECM dynamics can result from changes of ECM composition, for example, because of altered synthesis or degradation of one or more ECM components, or in architecture because of altered organization. Mounting evidence has shown how individual ECM components are laid down, cross-linked, and organized together via covalent and noncovalent modifications and how they can greatly influence the fundamental aspects of cell behavior (Lopez et al. 2008; Engler et al. 2009; Egeblad et al. 2010b). This higher level of ECM organization is also dynamic and subject to sustained remodeling as mediated by reciprocal interactions between the ECM and its resident cellular components (Daley et al. 2008). Understandably, ECM dynamics are tightly regulated to ensure normal development, physiology, and robustness of organ systems. This is achieved by redundant mechanisms to modulate the expression and function of ECM modifying enzymes at multiple levels. When such control mechanisms are corrupted, ECM dynamics become deregulated, leading to various human congenital defects and diseases, including cancer.Here, we examine the players involved in ECM remodeling and how they are tightly regulated to achieve a delicate balance between stability and remodeling of the ECM. We focus on the cellular and molecular mechanisms through which ECM dynamics influence cellular behaviors. We illustrate how a wide variety of cell behaviors can be deployed by exploiting the important roles of ECM dynamics to build vertebrate organs and maintain their functions, and how deregulation of ECM dynamics contributes to the initiation and progression of human cancer.     

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

HU (Histone‐like protein from Escherichia coli strain U93) is the most conserved nucleoid‐associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single‐molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface‐exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over‐condensed and mis‐segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid‐associated naRNAs, each previously implicated in HU’s high‐affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure‐binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.     

Calcium tips the balance: a microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4

Microtubules (MTs) are integral to numerous cellular functions, such as cell adhesion, differentiation and intracellular transport. Their dynamics are largely controlled by diverse MT‐interacting proteins, but the signalling mechanisms that regulate these interactions remain elusive. In this report, we identify a rapid, calcium‐regulated switch between MT plus end interaction and lattice binding within the carboxyl terminus of BPAG1n4. This switch is EF‐hand dependent, and mutations of the EF‐hands abolish this dynamic behaviour. Our study thus uncovers a new, calcium‐dependent regulatory mechanism for a spectraplakin, BPAG1n4, at the MT plus end.     

MicroRNAs in melanocyte and melanoma biology

The importance of microRNAs as key molecular components of cellular processes is now being recognized. Recent reports have shown that microRNAs regulate processes as diverse as protein expression and nuclear functions inside cells and are able to signal extracellularly, delivered via exosomes, to influence cell fate at a distance. The versatility of microRNAs as molecular tools inspires the design of novel strategies to control gene expression, protein stability, DNA repair and chromatin accessibility that may prove very useful for therapeutic approaches due to the extensive manageability of these small molecules. However, we still lack a comprehensive understanding of the microRNA network and its interactions with the other layers of regulatory elements in cellular and extracellular functions. This knowledge may be necessary before we exploit microRNA versatility in therapeutic settings. To identify rules of interactions between microRNAs and other regulatory systems, we begin by reviewing microRNA activities in a single cell type: the melanocyte, from development to disease.     

Flux an important, but neglected, component of functional genomics

Genomics approaches aimed at understanding metabolism currently tend to involve mainly expression profiling, although proteomics and steady-state metabolite profiling are increasingly being carried out as alternative strategies. These approaches provide rich information on the inventory of the cell. It is, however, of growing importance that such approaches are augmented by sophisticated integrative analyses and a higher-level understanding of cellular dynamics to provide insights into mechanisms that underlie biological processes. We argue the need for, and discuss theoretical and practical aspects of, the determination of metabolic flux as a component of functional genomics.